FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with United States Government support under grant no. NCT00549848 from the National Cancer Institute. The United States Government has certain rights in this invention.

RECOGNITION OF RESEARCH FUNDING

This invention was supported by funds received from the American Lebanese Syrian Associated Charities (ALSAC). REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 454638SEQLIST.txt, created on January 8, 2015, and having a size of 21 Kb and is filed concurrently with the

specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the detection of minimal residual disease in patients with acute lymphoblastic leukemia (ALL).

BACKGROUND OF THE INVENTION

Leukemia relapse is the major cause of treatment failure for patients with acute lymphoblastic leukemia (ALL) (Pui etal. (2009) N Engl J Med. 360:2730-2741 ; Gokbuget and Hoelzer (2009) Semin.Hematol. 46:64-75; and Faderl etal. (20 0) Cancer. 116:1165- 1176). Relapse originates from leukemic cells that are resistant to chemotherapy but become undetectable after initial treatment in most cases. Nevertheless, methods more sensitive than microscopic examination can demonstrate leukemic cells in a proportion of samples with no morphologic evidence of leukemia, a finding termed "minimal residual disease (MRD)"(Campana (2009) Hematol. Oncol Clin North Am. 23: 1083-98, vii).

MRD is currently the most powerful prognostic indicator in childhood ALL (Cave et al. (1998) N Engl J Med. 339:591-598; Coustan-Smith et al. (1998) Lancet. 351 :550-554; van Dongen et al. (1998) Lancet. 352: 1731-1738; Coustan-Smith et al. (2000) Blood. 96:2691-2696; Dworzak et al. (2002) Blood. 99: 1952-1958; Nyvold er a/. (2002) Blood. 99:1253-1258; Zhou et al. (2007) Blood. 1 10:1607-161 1 ; Borowitz et al. (2008) Blood. 1 11 :5477-5485. Basso et al. (2009) J Clin Oncol. 27:5168-5174; Conter et al. (2010) β/oocf. 1 15:3206-3214; Stow et al. (2010) Blood. 1 15:4657-4663). There is strong evidence supporting its prognostic significance in adult ALL (Krampera et al. (2003) Br J Haematol. 120:74-79; Vidriales , er a/. (2003) Blood. 101 :4695-4700; Raff er a/. (2007) Blood. 109:910-915; Holowiecki et al. (2008) Br. J. Haematol. 142:227-237; Bassan er a/. (2009) Blood. 1 13:4153-4162).

Thus, MRD monitoring has been introduced into many contemporary treatment protocols for risk assignment and selection of therapeutic regimens(Pui et al. (2009) N Engl J Med. 360:2730-2741 ; Gokbuget and Hoelzer. (2009) Semin. Hematol. 46:64-75; and Faderl er a/. (2010) Cancer. 116:1165-1176). MRD measurements are also clinically useful in patients with relapsed ALL who achieve a second remission (Coustan-Smith er al. (2004) Leukemia 18:499-504; Paganin er a/. (2008) Leukemia. 22:2193-2200; Raetz et al. (2008) J Clin. Oncol. 26:3971 -3978), can help optimize the timing of hematopoietic stem cell transplantation (Bader et al. (2009) J Clin Oncol. 27:377-384), and guide decisions about donor lymphocyte infusion post-transplant (Lankester er a/. (2010) Leukemia. 24:1462-1469).

Therefore, the identification of new markers for minimal residual disease is needed.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions are provided for predicting minimal residual disease (MRD) before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL). Compositions include an assay system comprising a plurality of probes for determining the expression levels of a panel of markers in a biological sample, wherein each of the probes specifically binds to a distinct marker, and wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, a fourth probe specifically binds to NKp46; and/or at least one probe capable of detecting a KIR2DL5A genotype; and, a system for predicting a MRD positive probability before induction chemotherapy comprising a database having a scoring criteria to predict the MRD positive probability. Further provided are kits for predicting MRD before induction chemotherapy in a subject comprising a plurality of probes for determining the expression levels of a panel of markers in a biological sample, wherein each of the probes specifically binds to a distinct marker, wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and/or a fourth probe specifically binds to NKp46. The kit can further include at least one probe capable of detecting a KIR2DL5A genotype.

Various methods of employing such compositions to predict MRD before induction chemotherapy in a subject having ALL are also provided, as are methods for generating a prognostic test to predict a MRD positive probability before induction chemotherapy in a subject having ALL.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows simplified maps of the A and B KIR haplotypes on chromosome 19q13.4. Cen-B is defined as KIR2DL2 positive and KIR2DL3 negative in the

centromeric motifs and Tel-B as KIR3DS1 positive and KIR3DL1 negative in the telomeric motifs.

Figure 2 shows the five biomarkers were used to establish predictive models either singly or as a 5-marker composite. The composite model has a 100% sensitivity (or 100% true positivity) and an 80% specificity (or 20% false positivity).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal

requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. /. Overview

Methods and compositions are provided for predicting minimal residual disease (MRD) before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL). As discussed in further detail herein, post-induction MRD has been established to be one of the most important prognostic markers for childhood ALL and MRD-based risk- adaptive continuation regimens have been shown to improve patient outcomes. Using five biomarkers associated with the NK-specific pathways discussed herein, a novel MRD-relevant predictive model has been developed that can be used before induction chemotherapy in a patient diagnosed with ALL so that risk-adaptive therapy can be implemented before (rather than after) induction chemotherapy.

II. Minimal Residual Disease (MRD) Positive Probability

Methods and compositions are provided herein directed to predicting MRD before induction chemotherapy in a subject having ALL. Leukemia is a cancer of the bone marrow and blood. The four major types of leukemia are acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Acute leukemia is a rapidly progressing cancer that produces blood cells that are not fully developed. Acute lymphocytic leukemia is often referred to as "acute lymphoblastic leukemia" because the leukemic cell that replace the normal marrow cell is the (leukemic) lymphoblast. There are two principal ALL subtypes including a) the B-lymphocyte subtype - identified by finding cell surface markers on the leukemic blast cells common to normal B lymphocytes; and b) the T lymphocyte subtypes - identified by finding cell surface markers on the leukemic blast cells common to normal T lymphocytes.

The effects of ALL include uncontrolled and exaggerated growth and

accumulation of lymphoblasts which fail to function as normal blood cells, and blockage of the production of normal marrow cells. The lack of production of normal marrow cells often leads to a deficiency of red cells (anemia), platelets (thrombocytopenia) and normal white cells, especially neutrophils (neutropenia), in the blood. ALL progresses rapidly without treatment, therefore the methods and compositions provided herein can be employed to allow for the early prognosis of minimal residual disease and thereby allow for the design of an appropriate treatment plan. In specific embodiments, following early prognosis of MRD positive probability, a risk adaptive therapy can be implemented either before, simultaneous with, during or after induction chemotherapy.

Most ALL patients achieve at least an initial remission. However, some patients have residual leukemic cells in their marrow. Other patients achieve remission then "relapse" wherein they have a decrease in normal blood cells and a return of leukemia cells in the marrow. Minimal residual disease (MRD) is the name given to small numbers of leukemic cells that remain in the patient during treatment, or after treatment when the patient is in remission (no symptoms or signs of disease). MRD is the major cause of relapse in cancer and leukemia.

The methods and compositions provided herein can predict a MRD positive probability prior to the administration of induction chemotherapy in a subject with ALL. Such methods and compositions thereby allow for a prognosis to be made earlier in the disease state and treatment protocols can thereby be adjusted according to the MRD positive probability. As used herein, an "MRD positive probability" is defined as the probability of positive MRD at the end of induction therapy. As summarized in Table 2, the MRD positive probability can be categorized into risk categories, such as, low risk, intermediate risk, or high risk. Based on the probability of positive MRD, the likelihood or risk of minimal residual disease (MRD) developing at the end of induction chemotherapy can be determined before induction chemotherapy is even administered to the subject having ALL. Such information can further help to evaluate treatment regimens, and in specific embodiments, allows for the design and implementation of appropriate risk- adaptive therapies. For example, the MRD positive probability can be indicative of the efficacy of certain treatment regimes, e.g., stem cell transplant. Methods for determining MRD positive probability are discussed elsewhere herein and in the experimental section.

Once the MRD positive probability is determined, one of skill in the art can determine the appropriate therapy to administer to the subject. As used herein, the term "therapy" can include any therapy for treating ALL, including but not limited to induction chemotherapy, chemotherapy, radiation therapy, stem cell transplantation, and biological therapy (e.g., monoclonal antibody therapy). Depending on the MRD positive probability, specific drugs or drug combinations, drug dosages, duration of treatment, and other types of treatment, may be indicated to achieve optimal results. For instance, patients in groups 1-16 as listed in Table 2 had a <1x10 ^"5 % risk of positive MRD; these patients would have a favorable outcome when treated with regimens similar to that of TotalXVI. By contrast, alternative therapy should be considered for patients in groups 17-25 of Table 2 who had an intermediate risk ranging from 0.2% to 10%, and especially for those in the high risk groups with a risk ranging from 13.3% to 81.6%. In specific embodiments, the predictive model can be implemented at diagnosis with favorable operating

characteristics: 100% sensitivity and 80% specificity, corresponding with a 100%) negative predictive value.

Methods and compositions are provided which comprise predicting the probability of minimal residual disease (MRD) before induction chemotherapy in a biological sample from a subject having acute lymphoblastic leukemia (ALL) by detecting the expression of a plurality of markers. The method can employ a plurality of probes wherein each of the probes specifically binds to a distinct marker and wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46; wherein each of the first, second, third and/or fourth probe is specific for determining the expression levels of the markers in a biological sample and, further comprising at least one probe capable of detecting the presence or the absence of the KIR2DL5A genotype.

In further embodiments, the first, second, third and fourth probe comprise an antibody or an antibody fragment. For example, the first probe can comprise a PI-9 antibody encoded by clone 7D8, the second probe can comprise a FasL antibody encoded by clone 14C2, the third probe can comprise a Granzyme B antibody encoded by clone GB11 , and the fourth probe can comprise an NKp46 antibody encoded by clone BAB281. When employing these specific antibodies, the criteria to predict the MRD positive probability that is employed is set forth in Table 2.

///. Markers for Predicting Minimal Residual Disease Positive Probability

As used herein a "marker" can be any gene or protein whose presence/absence or whose level of expression in a tissue or cell is used comparatively to evaluate the disease state of a given tissue or cell. In particular embodiments, antibodies are used to detect markers at the protein level. In other aspects, markers are detected at the nucleic acid level.

Markers may be referred to herein interchangeably as "markers," "MRD positive probability-associated phenotypic markers," "phenotypic markers," or "cell markers." "MRD positive probability-associated markers" can refer to particular combinations of markers used to predict MRD positive probability before induction chemotherapy in ALL patients. In particular embodiments, markers can refer to "antigenic markers," "antigens," or "cell surface antigens," referring to proteins that are expressed on the cell surface or RNA encoding the same, or markers can refer to genetic markers found in the genomic DNA. Various combinations of markers are provided that are selective for predicting MRD positive probability before induction chemotherapy in a subject having ALL. The various MRD positive probability-associated markers are set forth in Table 1.

Some of the markers employed in the methods and compositions disclosed herein can have a modulated level of expression when compared to an appropriate control, while other markers need only be present or absent from a given biological sample. The specific profiles of the given marker combinations that are predictive of minimal residual disease positive probability before induction chemotherapy in a subject having ALL are discussed in further detail elsewhere herein. Markers involved in the various methods and compositions provided herein include expression levels of PI-9, FasL, Granzyme B, NKp46, and the presence or absence of the KIR2DL5A genotype. Each of these markers, as well as, methods for detection of the markers are discussed in detailed elsewhere herein, and a brief summary is provided in Table 1.

Table 1. MRD Positive Probability-Associated Markers

Briefly, proteinase inhibitor-9 (PI-9, also designated cytoplasmic antiproteinase 3, or CAP3) is a member of the Ovalbumin family of serpins that is expressed in placenta, lung and cytotoxic lymphocytes. PI-9 is a potent inhibitor of granzyme B and of granzyme B-mediated apoptosis, and is also an inhibitor of caspase-1 and, to a lesser extent, caspase-4 and caspase-8. Because granzyme B promotes DNA degradation and rapidly translocates to the nucleus to bind to a nuclear component, PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells and epithelial cells. PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, suggesting that the nucleocytoplasmic distribution of PI-9 involves a nonconventional nuclear import pathway and the export factor CRM1. Estrogen rapidly and strongly induces PI-9, which is an estrogen-regulated human gene. PI-9 expression is also upregulated in response to inflammatory stimuli. This upregulation protects cells from apoptosis induced by endogenously expressed or released granzyme B, particularly during target cell killing. In addition, PI-9 is expressed in a variety of human and murine tumors. The PI-9 polynucleotide is found in NCBI accession No. NM_004155 and the amino acid sequence is set forth in NCBI accession No. P50453, each of which is herein incorporated by reference.

FasL is a member of the TNF-receptor superfamily and comprises a receptor containing a death domain. The interaction of this receptor with its ligand allows the formation of a death-inducing signaling complex that includes Fas-associated death domain protein (FADD), caspase 8, and caspase 10. The autoproteolytic processing of the caspases in the complex triggers a downstream caspase cascade, and leads to apoptosis. This receptor has been also shown to activate NF-kappaB, MAPK3/ERK1 , and MAPK8/JNK, and is found to be involved in transducing the proliferating signals in normal diploid fibroblast and T cells. The FasL sequence is set forth in UniProt Number P25445, which is herein incorporate by reference in its entirety. Alternative names for FasL include, for example, FAS, CD95, FAS1 , FAIM2, OTTHUMP00000059646, LFG, APT1 , FASTM, NMP35, APO-1 , ALPS1A, and TNFRSF6.

Granzyme B is a 32 kD serine protease, also known as granzyme-2, serine protease B, CCP1 , Asp-ase, and CTLA-1 . Granzyme B is abundantly stored in the granules of cytotoxic T lymphocytes and NK cells. Low level of expression has been reported in granulocytes, B cells, and activated dendritic cells. Granzyme B is needed for rapid induction of cell death and apoptosis through interaction with mannose-6-phosphate receptor. The sequence is set forth in NCBI Gene ID No. 3002, 14939, and 171528, each of which is herein incorporated by reference.

NKp46 is a 46-kDa transmembrane glycoprotein which is a NK-specific triggering receptor involved in non-MHC-restricted natural cytotoxicity and considered the prototype of the NK natural cytotoxicity receptors (NCRs). Its expression is restricted to all resting and activated NK cells, including the minor CD3 ^" CD56 ^bright CD16 ^" subset. Although NKp46 is highly expressed at the NK-cell surface in the majority of donors, some individuals have a proportion of NK cell (which vary up to 90%) expressing a "dull" NKp46 phenotype. NKp46 is a member of the immunoglobulin superfamily characterized by two extracellular C2-type Ig-like domains. It is associated with the ITAM bearing molecules ΟΌ3ζ and FcsRIy. Although this association is likely to be essential for signal transduction via NKp46, ΟΌ3ζ is not required for NKp46 surface expression. NKp46 represents a major activating receptor and plays a central role in the lysis and clearance of HLA class cells. The magnitude of lysis correlates with its level of surface expression. Other names for NKp46 include, for example, CD335, NCR1 , and natural cytotoxicity triggering receptor 1 . The sequence of NKp46 is set forth in, for example, NCBI Accession Nos. NM_004829 and NP_004820.1 076036, each of which is herein incorporated by reference.

Another marker for MRD positive probability comprises the KIR2DL5A genotype. Killer Immunoglobulin-like Receptors (KIR) comprise a family of membrane glycoproteins expressed on the cell surface of natural killer cells. KIR have specificity for Class I HLA molecules and the receptor-ligand interactions can either activate or inhibit cellular functions. In humans, there are 14 KIR genes and two pseudogenes located in the leukocyte receptor complex (LRC) on chromosome 19q13.4. Human NK cells express various combinations of these 16 KIR genes with two common haplotypes: Group A, which has more inhibitory receptors and Group B, which has more activating receptors. As demonstrated herein, the KIR2DL5A genotype comprises a MRD positive probability marker which can be used within the various methods, kits and assay systems to predict the minimal residual disease positive probability in a subject before induction

chemotherapy and having been diagnosed with ALL.

"Genotype" refers to the genetic constitution of a cell or organism. As used herein, the "KIR2DL5A genotype" comprises the genomic sequence of KIR2DL5A including both the coding region and/or any regulatory regions. The coding region of KIR2DL5A is set forth in SEQ ID NO: 1 and 2 and can also be found as EMBL accession no. ABM92655.1 and EMBL accession no. AFV74773.1 , both of which are herein incorporated by reference. The genomic DNA comprising KIR2DL5A is set forth in SEQ ID NO:3 with nucleotides 1422 to 10886 representing the KIR2DL5A coding region. It is further recognized that a KIR2DL5A genotype can be detected at the genomic level, the RNA level or at the protein level.

Various methods and compositions for identifying a KIR2DL5A genotype are provided. Such methods and compositions find use in identifying and/or detecting the KIR2DL5A genotype in any biological material, and the presence or absence of the KIR2DL5A genotype contributes to the marker panel provided herein for predicting minimal residual disease positive probability before induction chemotherapy in a subject having ALL.

In one embodiment, a method is provided for assaying a biological sample for the KIR2DL5A genotype. The method comprises (a) providing a biological sample from a subject, wherein the biological sample comprises the genomic DNA of the subject, and (b) determining if the genomic DNA comprises the KIR2DL5A genotype. In such a method, the presence or absence of the KIR2DL5A genotype contributes to the marker profile that allows for predicting minimal disease resistance positive probability before induction chemotherapy in a subject with ALL. Various methods of detecting the KIR2DL5A genotype are provided below.

The presence or absence of the KIR2DL5A genotype in combination with the expression level of the various MRD positive probability-associated markers discussed herein, including PI-9, FasL, Granzyme B, and/or NKp46, can be used in combination to predict the MRD positive probability before induction chemotherapy in a subject having ALL. a. Modulated Levels of MRD Positive Probability-Associated Markers

The various methods and compositions employ a plurality of antibodies, antibody fragments, or molecular probes wherein each antibody, antibody fragment, or molecular probe is specific for determining the expression levels of a panel of MRD positive probability-associated markers, such as PI-9, FasL, Granzyme B, and NKp46, in a biological sample, wherein each of the probes specifically binds to a distinct marker comprising PI-9, FasL, Granzyme B, and/or NKp46.

As used herein, a "modulated level" of a marker can comprise any statistically significant increase (overexpression) or decrease (underexpression) of the given marker when compared to an appropriate control. The modulated level can be assayed by monitoring either the concentration of and/or activity of the marker polypeptide and/or the level of the mRNA encoding the marker polypeptide. In general, a modulate level of marker can include either an increase or a decrease of at least at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher relative to an appropriate control.

By "overexpressed" it is intended that the marker of interest is overexpressed in a given biological sample but is not overexpressed in an appropriate control. By

"underexpressed" it is intended that the marker of interest is underexpressed in a biological sample and is not underexpressed in an appropriate control sample.

The level of expression of a particular marker that is sufficient to constitute "overexpression" will vary depending on the specific marker used. In particular embodiments, a "threshold level" or "cutoff value" of expression over a normal control is established for a particular marker, wherein expression levels above this value are deemed overexpression. Overexpression of a particular marker can refer to an increase in the percentage of a population detected as expressing a particular marker or marker combination. Overexpression can also refer to the level of expression on a population of cells as detected by an increase in the mean fluorescence intensity (MFI). In other embodiments, an overexpressed marker can include any statistically significant increase in expression when compared to an appropriate control, including for example, an increase of at least at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher relative to an appropriate control or at least at least a 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 10 fold or higher expression level relative to an appropriate control.

The level of expression of a particular marker that is sufficient to constitute "underexpression" will vary depending on the specific marker used. In particular embodiments, a "threshold level" or "cutoff value" of expression is established for a particular marker, wherein expression levels below this value are deemed

underexpression. Underexpression of a particular marker can refer to a decrease in the percentage of a population detected as expressing a particular marker or marker combination. Underexpression can also refer to the level of expression on a population of cells as detected by a decrease in the mean fluorescence intensity (MFI). In other embodiments, an underexpressed marker can include any statistically significant decrease in expression when compared to an appropriate control, including for example, a decrease of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or lower relative to an appropriate control or at least at least a 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 10 fold or lower expression level relative to an appropriate control.

Methods by which cutoff values which are predictive of MRD positive probability are determined are discussed elsewherein herein. In instances where the MRD positive probability is determined employing a PI-9 antibody encoded by clone 7D8, a FasL antibody encoded by clone 14C2, a Granzyme B antibody encoded by clone GB11 , and a NKp46 antibody encoded by clone BAB281 , the cutoff values for each of the markers are shown in Table 2 below, where <= is less than or equal to; < is less than; > is greater than and >= is greater than and equal to. As demonstrated herein, when employing the panel of markers/antibodies employed in Table 2, the MRD positive probability ranges between 3.32x10 ^'9 % to 81.9% depending on the level of expression of PI-9 when detected by the PI-9 antibody clone 7D8, the level of expression of FasL when detected by the FasL antibody encoded by clone 14C2, the level of expression of Granzyme B when detected by the Granzyme B antibody encoded by clone GB11 , the level of the NKp46 antibody when detected by BAB281 and the presence or absence of the KIR2DL5A phenotype. Each of the 32 groups shown in Table 2 represents a unique combination of the level and/or presence of these markers and further provides the MRD positive probability for each of the corresponding groups. The method by which the cutoff values were generated are discussed in further detail in the experimental section.

Table 2.

Risk Group Blast NK NK NK NK Probability Category PI-9 MFI FasL Granzyme B NKp46 KIR2DL5A MRD positive

MFI MFI % genotype (%)

1 <=650 >415 > 4070 > 30 - 3.32 x 10 ^"a

2 <=650 > 415 > 4070 > 30 + 1.34 x 10 ^"b

3 <=650 <=415 > 4070 > 30 - 1.87 x 10 ^"B

4 > 650 > 415 > 4070 > 30 - 2.36 x 10 ^~8

5 <=650 > 415 > 4070 <=30 - 3.72 x 10 ^"B

6 <=650 <=415 > 4070 > 30 + 7.57 x 10 ^"8

7 > 650 > 415 > 4070 > 30 + 9.57 x 10 ^"a

8 > 650 <=415 > 4070 > 30 - 1.33 x 10 ^" '

O

9 <=650 > 415 > 4070 <=30 + 1.51 x 10 ^" '

10 <=650 <=415 > 4070 <=30 - 2.09 x 10 ^" '

11 > 650 > 415 > 4070 <=30 - 2.64 x 10 ^" '

12 > 650 <=415 > 4070 > 30 + 5.38 x 10 ^" '

13 <=650 <=415 > 4070 <=30 + 8.47 x 10 ^" '

14 > 650 > 415 > 4070 <=30 + 1.07 x 10*

15 > 650 <=415 > 4070 <=30 - 1.49 x 10*

16 > 650 <=415 > 4070 <=30 + 6.03 x 10*

17 <=650 > 415 <=4070 > 30 - 0.2

18 <=650 > 415 <=4070 > 30 + 1

<u 19 <=650 <=415 <=4070 > 30 - 1.4

20 > 650 > 415 <=4070 > 30 - 1.7

21 <=650 > 415 <=4070 <=30 - 2.7

22 <=650 <=415 <=4070 > 30 + 5.3 a 23 > 650 > 415 <=4070 > 30 + 6.6

24 > 650 <=415 <=4070 > 30 - 8.9

25 <=650 > 415 <=4070 <=30 + 9.9

26 <=650 <=415 <=4070 <=30 - 13.3

27 > 650 > 415 <=4070 <=30 - 16.2

28 > 650 <=415 <=4070 > 30 + 28.3

29 <=650 <=415 <=4070 <=30 + 38.3

30 > 650 > 415 <=4070 <=30 + 44

31 > 650 <=4 5 <=4070 <=30 - 52.2

32 > 650 <=415 <=4070 <=30 + 81.6 b. Generating Expression Profiles

As used herein, an "expression profile" comprises one or more values

corresponding to a measurement of the relative abundance of a gene expression product (i.e., a marker). Such values may include measurements of RNA levels or protein abundance. Thus, an expression profile can comprise values representing the

measurement of the transcriptional state or the translational state of the gene. As is known to those of skill in the art, the transcriptional state and translational state are related.

In some embodiments, an "expression profile" of a biological sample can include the identities and relative abundance or "expression level" of the RNA species, especially mRNAs present in populations of cells in the biological sample. An expression profile can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies. For example, expression levels of RNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Patent Nos. 5,770,722,

5,874,219, 5,744,305, 5,677, 195 and 5,445,934, which are expressly incorporated herein by reference. Gene expression detection may also comprise nucleic acid probes in solution. Expression levels of RNA may also be monitored using the reverse

transcriptase polymerase chain reaction (e.g. , TaqMan®).

In one embodiment, microarrays are used to measure the values to be included in the expression profiles. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, U.S. Pat. Nos. 6,040, 138, 5,800,992 and 6,020, 135, 6,033,860, and

6,344,316, which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.

In one approach, total mRNA isolated from cells taken from the subject is converted to labeled cDNA and then hybridized to an oligonucleotide array. Each specimen is hybridized to a separate array. Relative transcript levels are calculated by reference to appropriate controls present on the array and in the sample. Embodiments can include, but are not limited to, the detection of mRNA expression with probes specific for polynucleotide encoding PI-9, FasL, Granzyme B, and/or NKp46. In specific embodiments, an expression profile is generated by the detection of nucleic acid corresponding to the expression of mRNA from a biological sample. A biological sample is contacted with a set of polynucleotides probes each of which specifically detects one of PI-9, FasL, Granzyme B, and/or NKp46 and determining the expression profile of each of said markers.

In other embodiments, an "expression profile" of a biological sample can include the identities and relative abundance or "expression level" of the constituent protein species expressed in populations of cells in the biological sample. Embodiments can include, but are not limited to, the detection of protein levels with antibody probes specific Pi-9, FasL, Granzyme B, and/or NKp46. In specific embodiments, a biological sample is contacted with a set of antibodies each of which specifically detects one of PI-9, FasL, Granzyme B, and/or NKp46 and determining the expression profile of each of said markers.

An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a prognostic and diagnostic tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve. ROC curves are well known to those skilled in the art. Methods of establishing such curves to generate prognostic tests to predict minimal residual disease positive probability employing any probe to the marker panel provided herein are discussed in further detail elsewhere herein.

The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cutoff selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

As discussed above, the measurement of the level of a single marker may have limited usefulness. In the methods and assay systems provided herein, data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to develop a panel of markers to provide a useful panel response. The data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file of any type. The database or data file may contain, for example, a patient identifier such as a name or number, the levels and/or presence or absence of the various relevant markers, and/or the MRD positive probability and/or the risk level of MRD before induction chemotherapy. The database can also include a database comprising detailed phenotypic information for a subject, medical history of the subject, family medical history and other lifestyle specific information for the subject. In another aspect, the assay system will include links to external databases to allow a user to communicate results of the diagnosis or prognosis to a medical practitioner for effecting suitable treatment for the subject.

An artificial cutoff region may be initially selected for each marker. The location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In one method, the cutoff region is initially centered about the center of the overlap region of the two sets of patients. In one embodiment, the cutoff region may simply be a cutoff point. In other embodiments, the cutoff region may have a length of greater than zero. In this regard, the cutoff region may be defined by a center value and a magnitude of length. In practice, the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range.

Each marker value for each patient may then be mapped to an indicator. The indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis. In other embodiments, the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.

The relative importance of the various markers may be indicated by a weighting factor. The weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In a preferred method, acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5. In a preferred embodiment, the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker.

Next, a panel response may be calculated for each subject in each of the two sets. The panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker. One advantage of using an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker. Typically, a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state. The use of an indicator which is constant on one side of the cutoff region eliminates this concern.

The panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient. Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker. The same marker may be measured again an hour later or some other time increment later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response.

Having obtained panel responses for each subject in each set of subjects, the distribution of the panel responses for each set may now be analyzed. An objective function may be defined to facilitate the selection of an effective panel. The objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.

In a one embodiment, the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function. For example, the objective function may reflect the area under the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers. In other

embodiments, other features of the ROC curve may be used to define the objective function. For example, the point at which the slope of the ROC curve is equal to one may be a useful feature. In other embodiments, the point at which the product of sensitivity and specificity is a maximum, sometimes referred to as the "knee," may be used. In an embodiment, the sensitivity at the knee may be maximized. In further embodiments, the sensitivity at a predetermined specificity level may be used to define the objective function. Other embodiments may use the specificity at a predetermined sensitivity level may be used. In still other embodiments, combinations of two or more of these ROC- curve features may be used.

It is possible that one of the markers in the panel is specific to the disease or condition being diagnosed. When such markers are present at above or below a certain threshold, the panel response may be set to return a "positive" test result. When the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function.

An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the art and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimums. In the optimization process, the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables. In a preferred embodiment, the weighting coefficient for each marker is also allowed to vary across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.

In addition to the above-described parameters, the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.

The optimization algorithm may be provided with certain constraints as well. For example, the resulting ROC curve may be constrained to provide an area-under-curve of greater than a particular value. ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets. Thus, a minimum acceptable value, such as 0.75, may be used as a constraint, particularly if the objective function does not incorporate the area under the curve. Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.

The iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function. The number of iterations may be limited in the optimization process. Further, the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum.

Thus, the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators. In order to develop lower-cost panels which require the

measurement of fewer marker levels, certain markers may be eliminated from the panel. In this regard, the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers. In one embodiment, the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated.

In certain cases, the lower weighting coefficients may not be indicative of a low importance. Similarly, a higher weighting coefficient may not be indicative of a high importance. For example, the optimization process may result in a high coefficient if the associated marker is irrelevant to the diagnosis. In this instance, there may not be any advantage that will drive the coefficient lower. Varying this coefficient may not affect the value of the objective function.

Individual panel response values may also be used as markers in the methods described herein. For example, a panel may be constructed from a plurality of markers, and each marker of the panel may be described by a function and a weighting factor to be applied to that marker (as determined by the methods described above). Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that particular marker to arrive at a sample value for that marker. The sample values for each marker are added together to arrive at the panel response for that particular sample to be tested. For a "diseased" and "non-diseased" group of patients, the resulting panel responses may be treated as if they were just levels of another disease marker.

One could use such a method to generate a prognostic test to predict minimal residual disease (MRD) positive probability before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL). The method comprises (1 ) obtaining a first biological sample from a first subject having ALL and positive for MRD at the end of induction chemotherapy and (2) obtaining a second biological sample from a second subject having ALL and negative for MRD at the end of induction chemotherapy. The first and the second biological sample are contacted with a plurality of probes, wherein each of the probes specifically binds to a distinct marker, wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46 and the complex formed between each of the probes with the markers is detected. A value is generated corresponding to an expression level of each of the marker in the first and the second biological sample. It is further determined if the genomic DNA in the first and the second biological sample comprises a KIR2DL5A genotype. A receiver operating characteristic (ROC) curve for each of the first, second, third, fourth probe, and for the KIR2DL5A genotype is generated for each of the first and the second samples by varying the cutoff values for each of the first, second, third, fourth probe, and for the KIR2DL5A genotype. An optimal point along each of the ROC curves generated is selected; and, the cutoff values corresponding to the optimal point of each ROC curve as the final values for the scoring criteria capable of predicting MRD positive probability before induction

chemotherapy are selected. c. Probes to Detect MRD Positive Probability-Associated Markers

The term "probe" refers to any molecule that is capable of specifically binding to an intended target molecule, for example, a nucleotide transcript or a protein encoded by a marker gene. RNA DNA probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Likewise, antibody probes to specific targets can be generated by one of skill in the art, or derived from appropriate sources. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. . Antibodies and Antibody Detection

In specific embodiments, the values in the expression profile are obtained by measuring the abundance of the protein products of the differentially-expressed genes. The abundance of these protein products can be determined, for example, using antibodies specific for the protein products of the differentially-expressed genes. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments, which can be generated by treating the antibody with an enzyme such as pepsin.

The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site (e.g., Fab', F'(ab) ₂ , Fv, single chain antibodies, diabodies).

Antibody derivatives may comprise a protein or chemical moiety conjugated to the antibody.

By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. An epitope is a site on an antigen or marker where the antibody binds via its variable region. The epitope is therefore a part of the antigen or marker, but the epitope is only a portion of the marker recognized by the antibody. According to this definition, an antibody is said to "specifically bind" to an epitope or have "antigen specificity" when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. As used herein, therefore, "specifically binds" is used interchangeably with recognition of a defined epitope on an antigen or marker, or any epitope contained in the antigen or marker. For example the term "specifically binds" when used in conjunction with a particular antibody is used to indicate that there is recognition of a certain epitope of the antigen and the interaction between the antibody and epitope is a non-random interaction indicative of the presence or "expression" of the certain epitope. The term "specifically binds" when used in conjunction with a particular marker is used to indicate that there is recognition of a certain antigen or marker and the interaction between the antibody and antigen or marker is a non-random interaction indicative of the presence or "expression" of the certain antigen or marker.

The antibody can be a polyclonal, monoclonal, or recombinant, e.g., a chimeric or humanized, fully human, non-human (e.g., murine, or single chain antibody). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.

The term "polyclonal antibody" as used herein refers to an antibody obtained from a population of heterogeneous antibodies derived from a multiple B cell response to an antigen which will recognize a variety of epitopes on the antigen. Polyclonal antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with a marker protein immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized biomarker protein. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, C. (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor, et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole, et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, NY), pp. 77-96) or trioma techniques. The technology for producing

hybridomas is well known (see generally Coligan, ei al. eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY); Galfre et al. (1977) Nature

266:550-52; Kenneth (1980) in Monoclonal Antibodies: A New Dimension In Biological Analyses (Plenum Publishing Corp., NY); and Lerner (1981 ) Yale J. Biol. Med., 54:387 402).

As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a marker protein to thereby isolate immunoglobulin library members that bind the marker protein. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01 ; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).

Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S.

Patent No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs ei al. (1991 ) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281 ; Griffiths et al. (1993) EMBO J. 12:725-734.

Antigen-binding fragments and variants of the monoclonal antibodies disclosed herein are contemplated and within the scope of the present invention. Such variants, for example, will retain the desired binding properties of the parent antibody. Methods for making antibody fragments and variants are generally available in the art. For example, amino acid sequence variants of a monoclonal antibody described herein can be prepared by mutations in the cloned DNA sequence encoding the antibody of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); U.S. Patent No. 4,873,192; and the references cited therein; herein incorporated by reference.

Guidance as to appropriate amino acid substitutions that do not affect biological activity of the polypeptide of interest may be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.

Preferably, variants of an antibody to a reference marker will have amino acid sequences that have at least 70% or 75% sequence identity, preferably at least 80% or 85% sequence identity, more preferably at least 90%, 91 %, 92%, 93%, 94% or 95% sequence identity to the amino acid sequence for the reference antibody molecule, or to a shorter portion of the reference antibody molecule. More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence identity. For purposes of the present invention, percent sequence identity is determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981 ) Adv. Appl. Math. 2:482-489. A variant may, for example, differ from the reference antibody by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

As discussed elsewhere herein, one or more of the antibodies, antibody fragments, or polynucleotide probes employed in the method can comprise a detectable label. Such detectable labels can comprise a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof.

The methods of the invention can comprise detection of the markers by flow cytometry with preferred combinations of probes to specific markers. Prediction of MRD positive probability can be combined with at least 4 different probes, and can include in some embodiments at least 5, 6, 7, 8, 9, 10, 11 , and 12 different probes.

Embodiments include methods, kits, and assay systems comprising combinations of probes to detect expression levels of PI-9, FasL, Granyzme B, and/or Nkp46.

In specific embodiments, methods, kits, and assay systems are provided which comprise (1) a PI-9 antibody encoded by clone 7D8, (2) a FasL antibody encoded by clone 14C2, (3) a Granzyme B antibody encoded by clone GB1 1 , and (4) a NKp46 antibody encoded by clone BAB281. Such methods, kits and assay systems can further employ the criteria to predict the MRD positive probability is set forth in Table 2. It is recognized that other antibodies for PI-9, FasL, Granzyme B, and NKp46 can be employed in the various methods and compositions disclosed herein. For example, additional antibodies that can be employed to detect NKp46 include A66902 from

Beckman Coulter and IHC-plus™ LS-B2105, LS-C187493, clone N1 D9 (LS-C93759), LS-C150015, LS-C120135, LS-C177862, LS-C44750, LS-C44749, LS-C151540, LS- C151539, LS-C44751 , LS-C16872, (clone 1 1 n142) LS-C184860, and LS-C131059 each from Lifespan Biosciences Inc.

Additional PI-9 antibodies include, for example, C-10, C-14, F-12, F-6, H-300, M- 244, 6D700, PI9-17, and 7D8 from Santa Cruz Biotechnology, Inc.

Additional Granzyme B antibodies include, for example, 10345-MM02, 10345-

R002, 10345-RP03, 10345-RP04 from Sino Biological Inc.

Additional FasL antibodies include, for example, BMS151 BT, BMS151 FI,

BMS151 , BMS245, BMS245CE, BMS1030, BMS138, BMS140BT, BMS140FI, BMS140, 53-0951 , 17-0951 , 13-0951 , and 12-0951 from Affymetrics eBioscience.

Antibodies which detect the KIR2DL5A genotype include, for example,

LS-C120055 and LS-C47477 from Lifespan Biosciences; 17-1588-41 and 17-1588-42 from eBioscience; and ABIN970999 from antibodies-online.com.

/ ^' /. Detection of Polynucleotides

As used herein, the use of the term "polynucleotide" is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and

ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

As used herein, the "nucleic acid complement" of a sample comprises any polynucleotide contained in the sample. The nucleic acid complement that is employed in the methods and compositions can include all of the polynucleotides contained in the sample or any fraction thereof. For example, the nucleic acid complement could comprise the genomic DNA and/or the mRNA and/or cDNAs of the given biological sample. Thus, the expression levels of any of the markers set forth in Table 1 can be detected through the level of the mRNA expression and the KIR2DL5A genotype can be detected in the genomic DNA, the RNA or cDNA or at the polypeptpide level.

As used herein, a "probe" is an isolated polynucleotide to which is attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, enzyme, etc. Such a probe is complementary to a strand of a target polynucleotide, which in specific embodiments of the invention comprise a polynucleotide that can specifically detect the KIR2DL5A genotype and/or one or more polynucleotide that can detect the level of expression of the various markers in Table 1. Deoxyribonucleic acid probes may include those generated by PCR using specific primers for the KIR2DL5A genotype (detection of the genomic DNA, cDNA or RNA) or the specific primers for mRNAs of the markers set forth in Table 1 , oligonucleotide probes synthesized in vitro, or DNA obtained from bacterial artificial chromosome, fosmid or cosmid libraries. Probes include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that can specifically detect the presence of the target DNA sequence. For nucleic acid probes, examples of detection reagents include, but are not limited to radiolabeled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), affinity labeled probes (biotin, avidin, or steptavidin), and fluorescent labeled probes (6-FAM, VIC, TAMRA, MGB, fluorescein, rhodamine, texas red [for BAC/fosmids]). One skilled in the art will readily recognize that the nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.

As used herein, "primers" are isolated polynucleotides that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs of the invention refer to their use for amplification of a target polynucleotide, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods. "PCR" or "polymerase chain reaction" is a technique used for the amplification of specific DNA segments (see, U.S. Pat. Nos. 4,683, 195 and 4,800,159; herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to the target DNA or mRNA or cDNA sequences and specifically detect and/or identify a polynucleotide comprising a KIR2DL5A genotype and/or the mRNA or cDNA of the markers set forth in Table 1. It is recognized that the hybridization conditions or reaction conditions can be determined by the operator to achieve this result. This length may be of any length that is of sufficient length to be useful in a detection method of choice. Generally, 8, 1 1 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700 nucleotides or more, or between about 1 1-20, 20-30, 30-40, 40-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or more nucleotides in length are used. Such probes and primers can hybridize specifically to a target sequence under high stringency hybridization conditions. Probes and primers according to embodiments of the present invention may have complete DNA sequence identity of contiguous nucleotides with the target sequence, although probes differing from the target DNA sequence and that retain the ability to specifically detect and/or identify a target DNA sequence may be designed by conventional methods. Accordingly, probes and primers can share about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity or complementarity to the target polynucleotide. Probes can be used as primers, but are generally designed to bind to the target DNA or RNA and are not used in an amplification process.

Specific primers can be used to amplify the KIR2DL5A genotype or the

mRNA/cDNA of the markers in Table 1 to produce an amplicon that can be used as a "specific probe" or can itself be detected for identifying an KIR2DL5A genotype in a biological sample and/or detect or identify the level of expression of the various markers in Table 1 in a biological sample. When the probe is hybridized with the polynucleotides of a biological sample under conditions which allow for the binding of the probe to the sample, this binding can be detected and thus allow for an indication of the presence of the KIR2DL5A genotype and/or the level of expression of the markers of Table 1 in the biological sample. Such identification of a bound probe has been described in the art. The specific probe may comprise a sequence of at least 80%, between 80 and 85%, between 85 and 90%, between 90 and 95%, and between 95 and 100% identical (or complementary) to a specific region of the KIR2DL5A genotype (genomic DNA, cDNA or mRNA) or the cDNA/mRNA of the markers of Table 1.

As used herein, "amplified DNA" or "amplicon" refers to the product of

polynucleotide amplification of a target polynucleotide that is part of a nucleic acid template. By "diagnostic" for a KIR2DL5A genotype is intended the use of any method or assay which discriminates between the presence or the absence of a KIR2DL5A genotype in a biological sample. By "diagnostic" for the expression level of the markers in Table 1 is intended the use of any method or assay which allows the level of expression of the given marker to be evaluated. The amplicon is of a length and has a sequence that is also diagnostic for the given purpose. The amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. A member of a primer pair derived from the flanking sequence may be located a distance from the junction or breakpoint. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol. 1-3, ed. Sambrook et a/., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 (hereinafter, "Sambrook et al., 1989"); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-lnterscience, New York, 1992 (with periodic updates) (hereinafter, "Ausubel et al., 1992"); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as the PCR primer analysis tool in Vector NTI version 10 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3 (Version 0.4.0.COPYRGT., 1991 , Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using guidelines known to one of skill in the art.

As outline in further detail below, any conventional nucleic acid hybridization or amplification or sequencing method can be used to specifically detect the presence of a polynucleotide. By "specifically detect" is intended that the polynucleotide can be used either as a primer to amplify the KIR2DL5A genotype or the polynucleotide can be used as a probe that hybridizes under stringent conditions to a polynucleotide comprising the KIR2DL5A genotype. The level or degree of hybridization which allows for the specific detection of the KIR2DL5A genotype is sufficient to distinguish the polynucleotide with the KIR2DL5A genotype from a polynucleotide that does not contain the KIR2DL5A genotype and thereby allow for discriminately identifying a KIR2DL5A genotype. By "shares sufficient sequence identity or complementarity to allow for the amplification" of a given target is intended the sequence shares at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or complementarity to a fragment or across the full length of the given target (i.e., the KIR2DL5A genotype or the mRNA/cDNA of any of the markers shown in Table 1 ).

The KIR2DL5A genotype may be detected and/or the expression level of the markers of Table 1 can be determined using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and, nucleic acid amplification. Nucleic acid hybridization includes methods using labeled probes directed against purified DNA, amplified DNA, and fixed leukemia cell preparations (fluorescence in situ hybridization).

Illustrative non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Additional methods for identifying nucleic acids containing KIR2DL5A genotype which do not necessarily require sequence amplification can also be employed and are based on, for example, the known methods of Southern (DNA: DNA) blot hybridizations, in situ hybridization and FISH of chromosomal material, using appropriate probes.

In situ hybridization (ISH) is a type of hybridization that uses a labeled

complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. The probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts. In some embodiments, the various markers of Table 1 are detected using fluorescence in situ hybridization (FISH).

In specific embodiments, probes for detecting KIR2DL5A genotype and/or the other markers listed in Table 1 are labeled with appropriate fluorescent or other markers and then used in hybridizations. The Examples section provided herein sets forth various protocols that are effective for detecting genomic abnormalities, but one of skill in the art will recognize that many variations of these assay can be used equally well. Specific protocols are well known in the art and can be readily adapted for the present invention. Guidance regarding methodology may be obtained from many references including: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In situ Hybridization: hi Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In situ Hybridization: A Practical Approach (ed. D. G.

Wilkinson), Oxford University Press Inc., England (1992)); Kuo et ai. (1991 ) Am. J. Hum. Genet. 42:1 12-1 19; Klinger ei a/. (1992) Am. J. Hum. Genet. 51 :55-65; and Ward et al. (1993) Am. J. Hum. Genet. 52:854-865). There are also kits that are commercially available and that provide protocols for performing FISH assays (available from e.g.,

Oncor, Inc., Gaithersburg, MD). Patents providing guidance on methodology include U.S. 5,225,326; 5,545,524; 6,121 ,489 and 6,573,043. All of these references are hereby incorporated by reference in their entirety and may be used along with similar references in the art and with the information provided in the Examples section herein to establish procedural steps convenient for a particular laboratory.

In hybridization techniques, all or part of a polynucleotide that selectively hybridizes to a target polynucleotide is employed. By "stringent conditions" or "stringent hybridization conditions" when referring to a polynucleotide probe is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are

100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of identity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length or less than 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is a polynucleotide that will specifically hybridize to the complement of the nucleic acid molecule to which it is being compared under high stringency conditions. Appropriate stringency conditions which promote DNA hybridization, for example, 6X sodium chloride/sodium citrate (SSC) at about 45° C, followed by a wash of 2X SSC at 50° C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. Typically, stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1 % SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5X to 1X SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1 % to about 1 % SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley- Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) and Haymes et al. (1985) In: Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, D.C.

A polynucleotide is said to be the "complement" of another polynucleotide if they exhibit complementarity. As used herein, molecules are said to exhibit "complete complementarity" when every nucleotide of one of the polynucleotide molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency" conditions. Similarly, the molecules are said to be "complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high-stringency" conditions.

Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook ei al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis ei al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).

Methods of amplification are further described in US Patent No. 4,683, 195, 4,683,202 and Chen ei al. (1994) PNAS 91 :5695-5699. These methods as well as other methods ^" known in the art of DNA amplification may be used in the practice of the embodiments of the present invention. It is understood that a number of parameters in a specific PCR protocol may need to be adjusted to specific laboratory conditions and may be slightly modified and yet allow for the collection of similar results. These adjustments will be apparent to a person skilled in the art.

The amplified polynucleotide (amplicon) can be of any length that allows for the detection of a given marker. For example, the amplicon can be about 10, 50, 100, 200, 300, 500, 700, 100, 2000, 3000, 4000, 5000 nucleotides in length or longer.

Thus, in specific embodiments, a method of detecting the presence of a

KIR2DL5A genotype in a biological sample is provided. The method comprises (a) providing a sample comprising the nucleic acid complement of a subject; (b) providing a pair of DNA primer molecules that can amplify an amplicon specific for a KIR2DL5A genotype (c) providing DNA amplification reaction conditions; (d) performing the DNA amplification reaction, thereby producing a DNA amplicon molecule; and (e) detecting the DNA amplicon molecule. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed. In one embodiment, the pair of DNA primer molecules amplify a piece of genomic DNA to detect the KIR2DL5A genotype. In another embodiment, the pair of DNA primer molecules amplify a cDNA or RNA to detect the KIR2DL5A genotype.

In still other embodiments, the various markers of Table 1 may be amplified prior to or simultaneous with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). The polymerase chain reaction (U.S. Pat. Nos. 4,683, 195, 4,683,202, 4,800,159 and 4,965, 188, each of which is herein incorporated by reference in its entirety), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. For other various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683, 195, 4,683,202 and

4,800, 159; Mullis et al, (1987) Meth. Enzymol. 155: 335; and, Murakawa et al., (1988) DNA 7: 287, each of which is herein incorporated by reference in its entirety.

Additional methods include the ligase chain reaction (Weiss (1991 ) Science 254:

1292, herein incorporated by reference in its entirety). Strand displacement amplification (Walker et al. (1992) Proc. Natl. Acad. Sci. USA 89: 392-396; U.S. Pat. Nos. 5,270,184 and 5,455, 166, each of which is herein incorporated by reference in its entirety), commonly referred to as SDA. See, for example, EP Pat. No. 0 684 315.

One illustrative detection method, the Hybridization Protection Assay (HPA) involves hybridizing a chemiluminescent oligonucleotide probe (e.g., an acridinium ester- labeled (AE) probe) to the target sequence, selectively hydrolyzing the chemiluminescent label present on unhybridized probe, and measuring the chemiluminescence produced from the remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283,174 and Nelson et al. (1995) Nonisotopic Probing, Blotting, and Sequencing, ch. 17 (Larry J.

Kricka ed., 2d ed., each of which is herein incorporated by reference in its entirety).

Another illustrative detection method provides for quantitative evaluation of the amplification process in real-time. Evaluation of an amplification process in "real-time" involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction, and using the determined values to calculate the amount of target sequence initially present in the sample. A variety of methods for determining the amount of initial target sequence present in a sample based on real-time amplification are well known in the art. These include methods disclosed in U.S. Pat. Nos. 6,303,305 and 6,541 ,205, each of which is herein incorporated by reference in its entirety. Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029, herein incorporated by reference in its entirety. Amplification products may be detected in real-time through the use of various self-hybridizing probes, most of which have a stem-loop structure. Such self-hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence. See, for example, U.S. Pat. No. 6,534,274, herein incorporated by reference in its entirety.

Another example of a detection probe having self-complementarity is a "molecular beacon." Molecular beacons include nucleic acid molecules having a target

complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation.

Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517 and 6, 150,097, herein incorporated by reference in its entirety.

Other self-hybridizing probes are well known to those of ordinary skill in the art. By way of non-limiting example, probe binding pairs having interacting labels, such as those disclosed in U.S. Pat. No. 5,928,862 (herein incorporated by reference in its entirety) might be adapted for use in the present invention. Probe systems used to detect single nucleotide polymorphisms (SNPs) might also be utilized in the present invention.

Additional detection systems include "molecular switches," as disclosed in U.S. Publ. No. 20050042638, herein incorporated by reference in its entirety. Other probes, such as those comprising intercalating dyes and/or fluorochromes, are also useful for detection of amplification products in the present invention. See, e.g., U.S. Pat. No. 5,814,447 (herein incorporated by reference in its entirety).

Additional methods that can be used to detect the markers of Table 1 include, but are not limited to, Genetic Bit Analysis (Nikiforov et al. (1994) Nucleic Acid Res. 22: 4167- 4175) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate. Following PCR of the region of interest (using one primer in the inserted sequence and one in the adjacent flanking sequence) a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization, and single base extension.

Another detection method is the Pyrosequencing technique as described by Winge ((2000) Innov. Pharma. Tech. 00: 18-24). Moreover, fluorescence Polarization as described by Chen et al. ((1999) Genome Res. 9: 492-498, 1999) is also a method that can be used to detect an amplicon of the invention, and Taqman® (PE Applied

Biosystems, Foster City, Calif.) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer.

/ ^' / ^' /. Optical Detection Methods

Detection of antibodies or polynucleotides can be facilitated by coupling (i.e., physically linking) the antibody or polynucleotide to a detectable substance (i.e., antibody labeling or polynucleotide labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials (fluorophores, flurochromes), luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of fluorophores/flurochromes, include phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin-chlorophyll (PerCP), allophycocyanin (APC), R-phycoerythrin conjugated with cyanine dye (PE-Cy7), allophycocyanin-cyanine tandem (APC-H7), coumarin dye (Horizon v450), sulphonyl chloride (Texas Red), cyanine (CY3, CY5, Cy7), FAM, JOE, TAMRA, TET, VIC, rhodamine; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵ l, ^{1 31} l, ³⁵ S or ³ H. The skilled artisan will understand that additional moieties may be suitable for the methods disclosed herein.

A detectable moiety generally refers to a composition or moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical or chemical means such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means. The terms "fluorophore" and "fluorochrome" are defined as a chemical group, or component of a molecule that causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. A fluorophore/fluorchrome can refer to various fluorescent substances, including dyes, used in fluorescence microscopy or flow cytometry to stain specimens. The terms fluorophore" and "fluorochrome" are herein used interchangeably.

Fluorochromes may be conjugated to antibodies, proteins, polypeptides, peptides, or nucleotide probes which specifically bind to antigens, proteins, polypeptides, peptides, polysaccharides, DNA, or RNA sequences. Thus, binding of an antibody, protein, polypeptide, peptide, or nucleotide probe to an antigen, protein, polypeptide, peptide, polysaccharide, DNA, or RNA may be detected by measuring a signal generated from a fluorochrome by flow cytometry, or any suitable optical imaging technique. Detection of a signal may indicate binding, whereas lack of detection of a signal may indicate lack of binding.

Methods and compositions for detectably labeling nucleic acid probes, such as oligonucleotides, DNA-RNA hybrids, etc. are well known in the art. See, e.g., U.S. Pat. Nos. 6,316,230; 6,297,016; 6,316,610; 6,060,240; 6,150,107; and 6,028,290, each of which is hereby incorporated by reference in their entirety.

An antibody or a fragment thereof can be conjugated with a detectable moiety, wherein the detectable moiety can be, for example, a fluorophore, a chromophore, a radionuclide, or an enzyme. In specific embodiments, a fluorophore can be, for example, phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin-chlorophyll (PerCP), allophycocyanin (APC), R-phycoerythrin conjugated with cyanine dye (PE-Cy7), allophycocyanin-cyanine tandem (APC-H7), and coumarin dye (Horizon v450). Detection of complexes formed between an antibody probe and marker can be achieved by an optical detection technique, including, but not limited to flow cytometry and microscopy.

"Cell staining" when used in reference to an antibody means that the antibody recognizes a marker and binds to a marker in the specimen forming a complex, thereby "labeling" or otherwise "staining" the cell expressing the marker to make it visible and/or detectable by microscopy or flow cytometry. Combinations of antibodies can be collectively added a specimen and thereby "stain the cell" for later analysis by

visualization with a flow cytometer or microscope, for example. One of skill in the art could determine whether a cell expressed a specific protein based on the level of antibody that bound to the cell using standard methods.

The methods disclosed herein can also be used in immunofluorescence histochemistry. This technique involves the use of antibodies labeled with various fluorophores to detect substances within a specimen. In exemplary embodiments a pathologist can derive a great deal of morphological information of diagnostic value by examining a specimen from a subject by microscope. Combinations of fluorophores or other detectable labels can be used by the methods on this invention, thereby greatly increasing the number of distinguishable signals in multicolor protocols. In another embodiment, the method employs flow cytometry. In another embodiment, in a peripheral blood sample or blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are used in one embodiment to exclude debris and dead cells.

Flow cytometry is an optical technique that analyzes particles or cells in a fluid mixture based on their optical characteristics, via the use of a flow cytometer (See, for example, Shapiro, "Practical Flow Cytometry," Third Ed. (Alan R. Liss, Inc., 1995); and Melamed et al. "Flow Cytometry and Sorting," Second Ed. (Wiley-Liss 1990)). Flow cytometers hydrodynamically focus a fluid suspension of particles/cells into a thin stream so that they flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles/cells.

Commonly used flow cytometers such as the Becton-Dickinson Immunocytometry Systems "FACSCAN" (San Jose, Calif.) can measure forward light scatter (generally correlated with the refractive index and size of the particle/cell being illuminated), side light scatter (generally correlated with the cell granularity), and particle fluorescence at one or more wavelengths. Data acquisition and analysis can be done using

FASCALIBER® LSRII flow cytometers (Becton Dickinson), and CELLQUEST Pro™, BD FACSDIVA™ software (both from Becton Dickinson), FLOWJO software (Tree Star, Ashland, OR) and/or KALUZA™ software (Beckman Coulter, Miami, FL)(Campana, D. (2009) Hematol Oncol Clin North Am. 23; 1083-98, vii).

In specific embodiments, determining an expression profile of a biological sample is generated using combinations of probes that bind specifically to binds to PI-9, FasL, Granzyme B, NKp46; and, determining the if KIR2DL5A genotype is present or absent.

In other embodiments, antibodies can be directly conjugated for simultaneous detection. For example, a method of the invention can comprise antibodies directly conjugated to a detectable fluorochrome for simultaneous detection of a plurality of markers. The skilled artisan will understand that any one antibody marker can be coupled to any fluorochrome for use in combination with any other antibody, and that preferred combinations can be used simultaneously with other antibody markers by the selection of different combinations of antibodies labeled with different flurochromes.

IV. Biological Samples

In some embodiments, the method comprises obtaining a "biological sample" or

"specimen" from a subject. The term "specimen" or "biological sample" is intended to include any clinically relevant tissue, such as, but not limited to, bone marrow samples, tumor biopsy, fine needle aspirate, or a sample of bodily fluid, such as, blood, plasma, serum, lymph, ascitic fluid, cystic fluid, urine, blood cells, bone marrow cells, and cellular products that are derived from blood and bone marrow cells. In specific embodiments, the biological sample comprises NK cells and leukemia blasts. Cellular products can include, but are not limited to, expressed proteins, expressed RNA, and DNA. In embodiments, a specimen can include cells derived from a variety of sources including, but not limited to, single cells, a collection of cells, tissue, cell culture, bone marrow, blood, or other bodily fluids. A tissue or cell source may include a tissue biopsy sample, a cell sorted population, cell culture, or a single cell. The term "biological sample" can be used interchangeably with the term "sample" or "patient sample."

A biological sample may be processed to release or otherwise make available a nucleic acid or a protein for detection as described herein. Such processing may include, for example, steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of RNA from the biological sample. Thus, the nucleic acid to be amplified in one embodiment by the methods of the invention may be DNA or RNA. Isolation of protein, RNA, and DNA from the aforementioned sources is known to those of skill in the art, and is discussed herein.

In one embodiment, the method comprises obtaining a peripheral blood sample from a subject and analyzing the expression level of specific markers in NK cells and leukemia blasts from the blood sample taken from the subject. To do blood tests, blood samples are generally taken from a vein in the subject's arm.

In another embodiment, the method comprises obtaining a bone marrow sample from a subject and analyzing the expression level of specific markers combinations in NK cells and leukemia blasts from the blood sample taken from the subject. Specimens of marrow cells are obtained by bone marrow aspiration and biopsy.

The obtaining of a biological sample uses methods well known in the art, as is the means to analyze NK cells and leukemia blasts populations. The method can be conducted on NK cells and leukemia blasts in blood samples which have not undergone any NK cells and/or leukemia blasts enrichment, on whole blood samples, or where red blood cells have been lysed. In other embodiments, the method can be conducted on enriched and purified subpopulations of cells (i.e., separate populations of NK cells and leukemia blasts) using methods well known in the art.

V. Analyzing Biological Samples

In specific embodiments, the method comprises "contacting" the biological sample with a plurality of probes. In one embodiment, the term "contacting" is in reference to probes that are antibodies and generally referring to methods of "cell staining." In one method, an antibody is added to a specimen and the antibody recognizes and binds to a specific protein for example, on the surface of cells in the specimen. A complex is thereby formed between the probe and the expressed protein. The complex can be detected and visualized by various techniques, as will be discussed herein.

Combinations of antibody probes can be collectively added to a biological sample and thereby "stain" the cell for later analysis by visualization with a flow cytometer or microscope, for example. One of skill in the art could determine whether a cell expressed a specific protein based on the level of antibody that bound to the cell using standard methods.

In other embodiments, the term "contacting" is used in reference to probes that are nucleic acids and refers to methods of detecting polynucleotides in a sample including the detection of an mRNA of interest in a biological sample and/or the detection of genomic DNA in a biological sample. A detectable complex can be formed when a nucleic acid probe specific to a gene of interest hybridizes and binds an mRNA/cDNA or genomic DNA in a biological sample. One of skill in the art could determine whether a cell expressed a specific mRNA or had a specific genomic DNA based on the level of detectable PCR product, for example, using standard methods.

VI. Cell Sorting and Selection of Subpopulations of Cells in a Biological Sample Multiparameter flow cytometric cell analysis can be used as part of the methods disclosed herein. The simultaneous analysis of multiple predictive parameters using flow cytometry is known to those of skill in the art. In one embodiment, the population of cells to be analyzed is contacted with a panel of antibodies directed against distinct cell surface markers, under conditions effective to allow antibody probe binding. The antibodies employed can be monoclonal antibodies, and can, in another embodiment, be labeled in a manner to allow their subsequent detection.

In other embodiments, fluorochromes can be excited by at least two different lasers to give off light of at least four different wavelengths, with the potential, for simultaneous analysis of at least four different markers. An additional two parameters include two light scattering parameters; direct and orthogonal, or side-scattering capability which can be analyzed concurrently with antibody detection, thereby allowing for cell analysis on the basis of at least 4 parameters. In further embodiments, at least five, six, seven, eight, nine, ten, eleven, or twelve different antibody probes, can be used simultaneously, thereby allowing for cell analysis on the basis of at least seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen different parameters.

Multiparameter cell sorting can be used in an embodiment to isolate cells based on a specific expression profile. For example, in one embodiment cell sorting analysis can be achieved using fluorescence-activated flow cytometry, by methods well described in the art.

As is well understood in the art and in yet other embodiments of the invention, various subsets of cells can be isolated for analysis, for example, it may be advantageous to compare NK cells and leukemia blasts. Enrichment of specific subpopulations of cells can be achieved by other methods as well. For example a wide variety of magnetic bead separation and isolation procedures can be used to selectively negatively and positively enrich samples for specific subpopulations of ceils. For example, in some embodiments a mixture of magnetic beads coupled to lineage specific antibodies can be used. The skilled artisan will understand that combinations of different antibodies can be used alone or in combination, and in multiple successive rounds of isolation, to positively and/or negatively select for subpopulations of cells.

One of skill in the art will recognize that optimization of reagents and conditions, for example, antibody titer and parameters for detection of antigen-antibody binding, is needed to maximize the signal to noise ratio for a particular antibody. Antibody concentrations that maximize specific binding to the markers of the invention and minimize non-specific binding (or "background") will be determined. In particular embodiments, appropriate antibody titers are determined by initially testing various antibody dilutions on patient serum samples. The design of assays to optimize antibody titer and detection conditions is standard and well within the routine capabilities of those of ordinary skill in the art. Some antibodies require additional optimization to reduce background and/or to increase specificity and sensitivity.

The skilled artisan will recognize that optimization of multiparameter assays designed to detect a plurality of antibody probes simultaneously will be necessary. In embodiments of the invention, maximization of signal to noise ratio, as well as

optimization of fluorochrome combinations will be necessary for each of the antibody probes combinations. Conjugated-antibody concentrations that maximize specific binding to the markers of the invention and minimize non-specific binding (or "background") will be determined with other such conjugated antibody probes as is known in the art. The design of assays to optimize and compensate the signals detected for the various conjugated antibodies is standard and well within the routine capabilities of those of ordinary skill in the art. Some antibodies require additional optimization to reduce background and/or to increase specificity and sensitivity.

VII. Kits and Assay Systems

Kits and assay systems for practicing the screening, prognostic, and diagnostic methods described herein are further provided. As used herein, "kit" refers to a set of reagents for the purpose of performing the various methods provided herein, more particularly, predicting minimal residual disease positive probability before induction chemotherapy in a biological sample from a subject having acute lymphoblastic leukemia. The term "kit" is intended to mean any manufacture (e.g., a package or a container) comprising at least one reagent, e.g., an antibody, a nucleic acid probe, etc. for specifically detecting the expression of a marker disclosed herein. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and methods for its use.

The kit can comprise a plurality of antibodies, antibody fragments, and/or molecular probes wherein each antibody, antibody fragment, or molecular probe is specific for determining the expression levels of a panel of markers in a biological sample, wherein each of the probes specifically binds to a distinct marker and wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46; and/or, at least one probe capable of detecting a KIR2DL5A genotype. In specific embodiments, the probe capable of detecting a KIR2DL5A genotype detects genomic DNA or detects cDNA or RNA. In other embodiments, the first, second, third and fourth probe comprises an antibody or an antibody fragment. For example, the first probe can comprise a PI-9 antibody encoded by clone 7D8, the second probe can comprise a FasL antibody encoded by clone 14C2, the third probe can comprise a Granzyme B antibody encoded by clone GB1 1 , and the fourth probe can comprise a NKp46 antibody encoded by clone BAB281. As discussed elsewhere herein, one or more of the antibodies, antibody fragments, or polynucleotide probes within the kit can comprise a detectable label. Such detectable labels can comprise a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof. The kit can further comprise instructions for use.

As used herein, an "assay system" refers to a set of reagents for the purpose of performing the method disclosed herein, more particularly, the reagents needed for the purpose of predicting minimal residual disease positive probability before induction chemotherapy in a biological sample from a subject having acute lymphoblastic leukemia and a corresponding system for predicting a MRD positive probability before induction chemotherapy. Such a system can comprise a database having a scoring criteria to predict the MRD positive probability. The assay system may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the assay system may contain a package insert describing the system and methods for its use.

In specific emboidments, the assay system comprises (1 ) a first probe comprising a PI-9 antibody encoded by clone 7D8, (2) a second probe comprising a FasL antibody encoded by clone 14C2, (3) a third probe comprising a Granzyme B antibody encoded by clone GB11 , and (4) a fourth probe comprising a NKp46 antibody encoded by clone BAB281 ; and, (5) scoring criteria to predict the MRD positive probability is set forth in Table 2. In further embodiments, the assay system comprises at least one probe capable of detecting a KIR2DL5A genotype. In still further embodiments, the probe capable of detecting a KIR2DL5A genotype comprise an antibody, a polynucleotide, a polynucleotide capable of detecting genomic DNA or a polynucleotide capable of detecting cDNA/mRNA.

One of skill in the art will further appreciate that any or all steps in the screening and diagnostic methods of the invention could be implemented by personnel or, alternatively, performed in an automated fashion. For example, the methods can be performed in an automated, semi-automated, or manual fashion, and as one-step or multi-step processes.

Non-limiting embodiments include:

1. An assay system for predicting minimal residual disease (MRD) before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL) comprising:

(a) a plurality of probes for determining the expression levels of a panel of markers in a biological sample, wherein each of said probes specifically binds to a distinct marker, wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46;

(b) at least one probe capable of detecting a KIR2DL5A genotype; and,

(c) a system for predicting a MRD positive probability before induction chemotherapy comprising a database having a scoring critera to predict the MRD positive probability.

2. The assay system of embodiment 1 , wherein said probe capable of detecting a KIR2DL5A genotype comprises a polynucleotide.

3. The assay system of embodiment 1 or 2, wherein said first, second, third and fourth probe comprise an antibody or an antibody fragment.

4. The assay system of embodiment 3, wherein

i) said first probe comprises a PI-9 antibody encoded by clone 7D8, the second probe comprises a FasL antibody encoded by clone 14C2, the third probe comprises a Granzyme B antibody encoded by clone GB11 , and the fourth probe comprises a NKp46 antibody encoded by clone BAB281 ; and,

ii) said scoring criteria to predict the MRD positive probability is set forth in

Table 2. 5. The assay system of any one of embodiments 2-4, wherein one or more of said antibodies, antibody fragments, or polynucleotide probes comprises a detectable label.

6. The assay system of embodiment 5, wherein said detectable label comprises a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof.

7. The assay system of any one of embodiments 1-6, wherein said database is further capable of storing information of the differential expression of the panel of markers of embodiment 1 (a) and the genotype of embodiment 1 (b) from the subject.

8. A kit for predicting minimal residual disease (MRD) before induction chemotherapy in a subject comprising:

(a) a plurality of probes for determining the expression levels of a panel of markers in a biological sample, wherein each of said probes specifically binds to a distinct marker, wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46; and,

(b) at least one probe capable of detecting a KIR2DL5A genotype.

9. The kit of embodiment 8, wherein said probe capable of detecting a KIR2DL5A genotype comprises a polynucleotide.

10. The kit of embodiment 8 or 9, wherein said first, second, third and fourth probe comprises an antibody or an antibody fragment.

11. The kit of embodiment 8, 9 or 10, wherein said first probe comprises a Pl- 9 antibody encoded by clone 7D8, the second probe comprises a FasL antibody encoded by clone 14C2, the third probe comprises a Granzyme B antibody encoded by clone GB11 , and the fourth probe comprises a NKp46 antibody encoded by clone BAB281.

12. The kit of any one of embodiments 8, 9, 10, or 11 , wherein one or more of said antibodies, antibody fragments, or polynucleotide probes comprises a detectable label.

13. The kit of embodiment 12, wherein said detectable label comprises a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof.

14. A method of predicting minimal residual disease (MRD) before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL) comprising:

(a) obtaining a biological sample from a subject diagnosed with ALL, wherein said subject has not undergone induction chemotherapy;

(b) contacting the biological sample with a plurality of probes, wherein each of said probes specifically binds to a distinct marker, wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46,

(c) detecting a complex formed between each of said probes in step (b) with said markers, wherein a value is generated corresponding to an expression level of each of said marker;

(e) determining if the genomic DNA in the biological sample comprises a KIR2DL5A genotype;

(f) generating a MRD positive probability profile by combining the values generated in step (c) and (e); and,

(g) comparing the MRD positive probability profile generated in (f) with a scoring criteria capable of predicting an MRD positive probability; and thereby predicting the likelihood of minimal residual disease (MRD) before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL).

15. The method of embodiment 14, wherein determining if the genomic DNA in the biological sample comprises a KIR2DL5A genotype comprises dectecting the

KIR2DL5A genotype with a polynucleotide probe that detects the genomic DNA.

16. The method of embodiment 15, wherein determining if the genomic DNA in the biological sample comprises a KIR2DL5A genotype comprises dectecting the

KIR2DL5A genotype with a polynucleotide probe that detects an RNA transcript or a cDNA.

17. The method of embodiment 14, 15, or 16, further comprising treating said subject with an appropriate therapy in view of the MRD positive probability.

18. The method of embodiment 14, 15, 16, or 17, wherein at least one of said first, second, third and fourth probe comprises an antibody or an antibody fragment.

19. The method of embodiment 18, wherein the antibody fragment comprises a F(ab') ₂ , Fab', Fv, Fd', or Fd fragment.

20. The method of embodiment 18, wherein

i) said first probe comprises a PI-9 antibody encoded by clone 7D8, the second probe comprise a FasL antibody encoded by clone 14C2, the third probe comprises a Granzyme B antibody encoded by clone GB1 1 , and the fourth probe comprises a NKp46 antibody encoded by clone BAB281 ; and,

ii) said scoring criteria capable of predicting MRD positive probability before induction chemotherapy is set forth in Table 2.

21. The method of any one of embodiments 14-20, wherein the biological sample is from a blood sample or a bone marrow sample.

22. The method of any one of embodiments 14-21 , wherein the at least one antibody is conjugated with a detectable moiety. 23. The method of embodiment 22, wherein the detectable moiety is a fluorophore, a chromophore, a radionucleotide, or an enzyme.

24. The method of embodiment 23, wherein at least one antibody is conjugated to a fluorophore comprising phycoerythrin (PE), fluorescein isothiocyanate (FITC), PerCP, APC, PE-Cy7, APC-H7, or Horizon v450.

25. The method of any one of embodiments 14-24, wherein detecting utilizes an optical detection technique.

26. The method of any one of embodiments 14-24, wherein said detecting utilizes flow cytometry.

27. A method for generating a prognostic test to predict a minimal residual disease (MRD) positive probability before induction chemotherapy in a subject having acute lymphoblastic leukemia (ALL) comprising:

a) obtaining a first biological sample from a first subject having ALL and positive for MRD at the end of induction chemotherapy and a second biological sample from a second subject having ALL and negative for MRD at the end of induction chemotherapy;

b) contacting the first and the second biological sample with a plurality of probes, wherein each of said probes specifically binds to a distinct marker, wherein a first probe specifically binds to PI-9, a second probe specifically binds to FasL, a third probe specifically binds to Granzyme B, and a fourth probe specifically binds to NKp46,

c) detecting a complex formed between each of said probes in step (b) with said markers, wherein a value is generated corresponding to an expression level of each of said marker in said first and said second biological sample;

d) determining if the genomic DNA in the first and the second biological sample comprises a KIR2DL5A genotype;

e) generating a receiver operating characteristic (ROC) curve for each of said first, said second, said third, said fourth probe, and for said KIR2DL5A genotype for each of said first and said second samples by varying the cutoff values for each of said first, said second, said third, said fourth probe, and for said KIR2DL5A genotype;

f) selecting an optimal point along each of the ROC curves generated in

(e); and,

g) selecting the cutoff values corresponding to the optimal point of each ROC curve as the final values for the scoring criteria capable of predicting MRD positive probability before induction chemotherapy.

28. The method of embodiment 27, wherein determining if the genomic DNA in the first and the second biological sample comprises a KIR2DL5A genotype comprises dectecting the KIR2DL5A genotype with a polynucleotide probe that detects the genomic DNA.

29. The method of embodiment 27, wherein determining if the genomic DNA in the biological sample comprises a KIR2DL5A genotype comprises dectecting the

KIR2DL5A genotype with a polynucleotide probe that detects an RNA transcript or a cDNA.

30. The method of embodiment 27, 28 or 29, wherein at least one of said first, second, third and fourth probe comprises an antibody or a fragment thereof.

31. The method of embodiment 30, wherein the antibody fragment comprises a F(ab') ₂ , Fab', Fv, Fd\ or Fd fragment.

32. The method of any one of embodiments 27-31 , wherein the biological sample is from a blood sample or a bone marrow sample.

33. The method of any one of embodiments 27-32, wherein the at least one antibody is conjugated with a detectable moiety.

34. The method of embodiment 33, wherein the detectable moiety is a fluorophore, a chromophore, a radionuclide, or an enzyme.

35. The method of embodiment 34, wherein at least one antibody is conjugated to a fluorophore comprising phycoerythrin (PE), fluorescein isothiocyanate (FITC), PerCP, APC, PE-Cy7, APC-H7, or Horizon v450.

36. The method of any one of embodiments 27-35, wherein detecting utilizes an optical detection technique.

37. The method of any one of embodiments 27-36, wherein said detecting utilizes flow cytometry.

The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element.

Throughout the specification the word "comprising," or variations such as

"comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Embodiments of the present invention are further defined in the following

Examples. It should be understood that these Examples are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

EXPERIMENTAL

Purpose

Not all natural killer (NK) cells are equally cytotoxic against leukemia because of differences in receptor gene content and surface expression. We correlated NK cell genotype and phenotype at diagnosis with minimal residual disease (MRD) after induction chemotherapy in childhood acute lymphoblastic leukemia (ALL), with a specific aim of developing a novel NK model to predict treatment response.

Patients and Methods

We analyzed the NK cells and leukemia blasts of 244 pediatric patients with ALL at diagnosis by killer-cell immunoglobulin-like receptor (KIR) typing and

immunophenotyping. The results were correlated statistically to post-induction MRD status.

Results

The odds of being MRD positive in patients with KIR telomeric {Tel)-A/B haplotypes was 2.85 fold of those with Tel-A/A haplotypes (p = 0.035). In comparison to the MRD negative group, patients with positive MRD were more likely to have KIR2DL5A (p=0.006) and expressed less activating receptor NKp46 and FASL on their NK cells (p=0.0074 and p=0.029, respectively). The concurrent expression of HLAC2 and

KIR2DL1 increased the odds of being MRD positive by 2.01 fold (p = 0.034). Additionally, the quantity of granzyme B inhibitor PI-9 in the leukemia blasts was greater in patients who were MRD positive (p=0.038). A simple 5-parameter model including NKp46, FASL, Granzyme B, PI-9, and Tel-B associated KIR2DL5A can predict MRD positivity at the end of induction with 100% sensitivity and 80% specificity.

Conclusion

NK cell biomarkers at diagnosis can predict response to induction chemotherapy in pediatric ALL. A strong NK effector phenotype is associated with better leukemia control.

INTRODUCTION

The natural killer (NK) cell is a lymphocyte important in cancer control ^1,2 . A diverse repertoire of cell surface inhibitory and activating receptors dictates the NK cell's response to both normal and abnormal cells. Once activated, the NK cell can lyse the target directly or secrete cytokines and chemokines that indirectly stimulate the host's adaptive immune response ³ .

NK cell cytotoxicity has been studied in hematological malignancies including acute myeloid leukemia (AML) ^4,5 and acute lymphoblastic leukemia (ALL) ^6"9 . Additionally, many have examined the best use of NK cells to achieve maximum benefit in

hematopoietic stem cell transplantation for AML or ALL ^10,11 . Due to differences in gene content and variable surface receptor expression, not all NK cells are alike, however ¹⁰ . We aimed to understand which NK cell biomarkers are most relevant in childhood ALL therapy.

Among several useful prognostic indicators for risk-directed therapy including National Cancer Institute-Rome Criteria based on presenting leukocyte count and age, genetic abnormalities, and immunophenotype, early response to treatment is the most predictive marker for the risk of relapse ¹² . It is because early response assessment by minimal residual disease (MRD) testing provides a precise and objective measurement of drug sensitivity of leukemia cells and the efficacy of treatment as well as host

pharmacogenetics and immune surveillance ¹⁷ .

In this study, we prospectively investigated the relationship between features of the NK cells at the time of ALL diagnosis and MRD at the end of induction chemotherapy to determine whether NK biomarkers can be utilized as prognostic marker before chemotherapy begins.

PATIENTS AND METHODS

Patients

Patients, 18 years old or younger, with newly diagnosed B- or T-lymphoblastic leukemia were enrolled on the institutional protocol Total Therapy Study XVI (TotalXVI). A total of 244 patients were enrolled from October, 2007 to October, 201 1 at the time this laboratory research was performed. These studies were approved by the institutional review board. Written informed consent was obtained from the guardians, and assent from the patient, as appropriate.

Remission Induction Therapy

All patients received intrathecal chemotherapy with methotrexate, cytarabine, and hydrocortisone on day 1 and 15. Patients with Philadelphia chromosome, MLL rearrangement, hypodiploidy (<44 chromosomes), or WBC count >100 x 10 /L at presentation received additional intrathecal treatment on days 8 and 22 and those with T cell ALL, t(1 ; 19)/7CF3-PSXf, CNS 2 status (<5 WBC/ML CSF with blasts), CNS 3 status (≥5 WBC/ L of CSF with blasts or cranial nerve palsy) or traumatic lumbar puncture (>10 RBC with blasts) received additional intrathecal treatment on days 4 and 1 1 . Induction treatment began with prednisone, vincristine, daunorubicin, and PEG asparaginase, followed by cyclophosphamide plus cytarabine and thioguanine. Dexamethasone was substituted for prednisone in patients with early T-cell precursor immunophenotype and mercaptopurine for thiopurine in those with thiopurine methyltransferase deficiency. An extra dose of PEG asparaginase was given if the day 15 MRD was≥ 1 % and fractionated cyclophosphamide at 300mg/m ² per dose every 12 hours for four doses was used instead of 1000mg/m ² for one dose if the day 15 MRD was >5%.

Assessment of response to induction therapy

Bone marrow aspirate for MRD determination was performed in all patients on day 38-42 of remission induction, when the absolute neutrophil count recovered to >300/μί, WBC >1000/μί, and platelets >50,000/μί. MRD was measured with flow cytometry or polymerase chain reaction (PCR) of immunoglobulin or T-cell receptor gene

rearrangement, as described previously. ¹⁷ MRD <0.01 % was considered negative.

NK cell and leukemia blasts immunophenotyping

Peripheral whole blood (3 ml) was obtained at diagnosis for immunophenotyping by flow cytometry of NK cells and leukemic blasts. The following antibodies were used to enumerate NK cells and their surface expression: anti-KIR2DL1 (14321 1 ), anti-KIR3DL1 (DX9), anti-KIR2DL2/3 (CH-L), anti-NKG2a (Z199), anti-CD1 1 a (HI 1 1 1 ), anti-NKG2D (1 D1 1 ), anti-TRAIL (RIK-2), anti-DNAM (DX1 1 ), anti-NKp30 (Z25), anti-NKp44 (Z231 ), anti-NKp46 (BAB281 ), anti-NTB-A (29281 1 ), anti-2B4 (2-69), anti-FAS (CIB2), anti-FASL (14C2), and anti-granzyme B (GB1 1 ). Blasts obtained at diagnosis were evaluated by anti-NTB-A (29281 1 ), anti-ULBP1 (170818), anti-ULBP2 (165903), anti-ULBP3 (166510), anti-ICAM (84H10), anti-Nectin (R2.525), anti-TRAIL-R1 (DJR1 ), anti-TRAIL-R2 (DJR2- 4), anti-MICA (159227), anti-MICB (23651 1 ), anti-PVR (TX21 ), anti-CD48 (J4-57), anti- H LA-ABC (W6/32), anti-PI-9 (7D8), and anti-FAS (CIB2).

KIR genotyping

PCR amplification was performed with the Olerup SSP KIR genotyping kit (Qiagen). The following gene alleles were tested: 2DL1 *001 -010, 2DL2*001 -005, 2DL3*001 -007, 2DL5A*0010101 -0010102 and 0050101 -005010102, 2DL5B*00201010- 004 and 00601 -009, 2DS1 *001 -004, 2DS2*0010101 -005, 2DS3*00101 -004,

2DS4*00101010-00103, 003, 004, 006, 007 and 009, 2DS5*001 -008, 3DL1 *00101 -002, 00401 -009, 01501-044, 056 and 057, 3DL2*00101-021 , 3DL3*00101-031 , 3DS1 *010- 014, 045-049N and 055, 2DP1 *00101-003, and 3DP1 *001 -006, 00301 -00301 and 004- 006. KIR2DL1 functional allele typing and KIR ligand typing were performed using a single nucleotide polymorphism (SNP) assay on the 7900 HT Sequence Detection System (Applied Biosystems) as described previously ¹³ .

NK cell receptor transcripts

Quantification of NK cell transcripts was performed for the following: KIR2DS1-5, KIR2DL1-4, KIR3DL1-2, KIR3DS1, NKp30, NKp44, NKp46, and NKG2D. Real-time quantitative PCR (RQ-PCR) was performed by the 7900 HT Fast Real-Time PCR system. cDNA was obtained by performing reverse transcription on RNA using the Invitrogen Vilo kit (Invitrogen, Carlsbad, CA, USA).

PCR reactions were performed with either 2.5 iL cDNA (from 12.5ng RNA), 2.5 uL of standards, or 2.5 uL of RNAse free water for the negative control. The master mix for all receptors, except KIR2DS3, included 12.5 μΙ_ of SYBR Green PCR master mix (Applied Biosystems), 2 \iL of forward and reverse primers at a concentration of 5 μΜ, and 6 pL of RNAse free water. Standards were diluted in EB buffer from 10 ⁵ copies down to 1 copy per 2.5μΙ_. Cycling parameters were as follows: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds, and 1 cycle at 72°C for 3 minutes. The dissociation steps were 95°C for 15 seconds, 60°C for 20 seconds, 95°C for 15 seconds.

The PCR reaction for KIR2DS3 was performed differently because the sensitivity of this receptor was not as high as the others. The master mix contained 10pL of Fast SYBR Green, 2μΙ_ of forward and reverse primer at a concentration of 5μΜ, and 3.5μΙ_ of RNAse and DNAse free water. Standards for KIR2DS3 were diluted in EB buffer from 10 ⁶ copies down to 10 copies per 2.5μΙ_. Cycling parameters were as follows: 95°C for 20 seconds, 40 cycles at 95°C for 1 second and 60°C for 20 seconds, and a hold at 72°C for three minutes. The dissociation steps were the same as the setup for all other receptors.

Forward and reverse primers for receptors 2DL1-4, 3DL2, 2DS1 -5, and 3DS1 are described previously ¹⁴ . Forward and reverse primers for the remaining receptors were as follows: KIR3DL1 5'- CAAGCTCCAAATCTGGTAACCC-3' (SEQ ID NO: 4) and 5'- CCAACTGTGCGTATGTCACC-3' (SEQ ID NO:5), NKp30 5'- CCCACTTGCTTCTTCCCGTTTCC-3' (SEQ ID NO: 6) and 5'- CACCACCAGCCGAGTCCCATTCC-3' (SEQ ID NO: 7), NKp44 5'- TCTCTAAGTCCGTCAGATTC-3' (SEQ ID NO: 8) and 5'- G ATG GTAG ATG G AG ACTCAG-3' (SEQ ID NO: 9), NKp46 5'- ACGGGACTCCAGAAAGACCAT-3' (SEQ ID NO: 10) and 5'- CAGGCCCATCCGAAGGA-3' (SEQ ID NO: 1 1) , and NKG2D 5'- GGCTCCATTCTCTCACCCA-3' (SEQ ID NO: 12) and 5'- TAAAGCTCGAGGCATAGAGTGC-3' (SEQ ID NO: 13) .

Statistical analysis

, We used the Fisher's exact test and Wilcoxon rank sum test to compare categorical and continuous baseline variables between patients with positive or negative MRD at the end of induction chemotherapy, respectively. Univariate logistic regression was used to test associations between KIR genotype, NK cell receptor surface expression, leukemia blast characteristics, KIR haplotypes and MRD status, respectively. Wilcoxon rank sum test was used for comparison of the NK cell receptor mRNA transcript level between patients with positive or negative MRD. Receiver operating characteristic (ROC) curves, area under the curve (AUC) of the ROC, sensitivity, and specificity were calculated to determine levels of PI-9, FasL, Granzyme B, and NKp46 that best differentiate positive MRD vs. negative MRD using the maximum Youden index method implemented in R package pROC ^15,16 . The smooth ROC curves were obtained using the method of maximum-likelihood fitting of univariate distributions (method = "fitdistr" in pROC). A scoring system based on the cutoffs of these four variables and the presence of KIR2DL5A, to predict MRD was developed using logistic regression model. All the reported p-values are 2-sided and are considered significant if <0.05 because of exploratory nature of the study. Statistical analyses were performed with R-2.15.1 ¹⁷

RESULTS

Table 3 shows the presenting clinical and biological features of the 244 patients studied and the distribution of these features according to the MRD status at the end of induction. Not surprisingly, the MRD negative group was younger than the MRD positive group (mean + SD, 6.86 ± 4.78 and 9.36 ± 4.71 years, respectively, p=0.0087).

Table 3. Patient Characteristics

Characteristics All patients MRD Negative MRD Positive p-value

Number of patients 244 217 27

Age at Diagnosis (yrs) 0.0087

Mean (SD) 7.14 (4.82) 6.86 (4.78) 9.36 (4.71 )

Median (Range) 5.44 (0.16-18.89) 5.26 (0.16-18.89) 9.71 (1.97-17.72)

Gender, N(%) 1

Female 106 (43.44) 94 (43.32) 12 (44.44)

Male 138 (56.56) 123 (56.68) 15 (55.56)

WBC at Diagnosis (10 ³ ) 0.506

Mean (SD) 39.41 (75.93) 38.59 (77.61 ) 46.01 (61.62)

Median (Range) 10.15 (0.5-591.5) 10.1 (0.5-591.5) 20 (1.4-246.5)

Immunophenotype N(%) 0.78

B-lineage 205 (84.02) 183 (84.33) 22 (81.48)

T-lineage 39 (15.98) 34 (15.67) 5 (18.52)

NK cell Genotypes

Table 4 shows the proportion of patients positive for each KIR gene according to their MRD status. The frequency distribution of positivity in the entire cohort was no different than the general United States population ¹⁸ . In a univariate logistic regression analysis, KIR2DL5A, KIR2DS1, and KIR2DS3 were statistically significant and were associated with increased odds of MRD by 3.05 to 4.5-fold. Notably, these three genes are found exclusively in the B haplotypes (Figure 1 ).

Table 4. Comparison of KIR genotype and MRD

% gene positive

KIR gene All patients Negative MRD Positive MRD OR (95%CI) p value

KIR2DL1 97.9 97.7 100 0(0-lnf) 0.99

KIR2DL2 53.7 54.7 44.4 0.66(0.25-1.76) 0.41

KIR2DL3 93.2 93 94.4 1.28(0.16-10.42) 0.82

KIR2DL5A 40 36.6 72.2 4.5(1.53-13.21) 0.006

KIR2DL5B 26.8 25.6 38.9 1.85(0.68-5.07) 0.23

KIR2DS1 31.6 29.1 55.6 3.05(1.14-8.18) 0.027

KIR2DS2 48.2 48.5 44.4 0.85(0.32-2.25) 0.74

KIR2DS3 24.2 21.5 50 3.65(1.35-9.85) 0.011

KIR2DS4*001 50 50 50 1 (0.38- 2.64) 1

KIR2DS4*003-9 74.7 74.4 77.8 1.2(0.38-3.85) 0.76

KIR2DS5 27.4 26.2 38.9 1.8 (0.66-4.92) 0.25

KIR3DL1 97.4 97.1 100 0(0-1 nf) 0.99

KIR3DS1 30 27.9 50 2.58(0.97-6.9) 0.058

KIR2DP1 97.9 97.7 too 0(0-lnf) 0.99

KIR3DL1 *004 25.4 25.7 22.2 0.82(0.26-2.64) 0.75

The patients' KIR genotypes were then categorized based on the presence of centromeric (Cen) or telomeric (Tel) A and B haplotype motifs as described previously ¹⁰ . The odds of being MRD positive in patients with Tel A/B was 2.85 times than those with Tel A/A (95% CI 1.075-7.73, p = 0.035). No statistically significant difference was found between Cen A/A and Cen A/B. The effect of Cen B/B and Tel B/B was not examined because of the small number of patients (<15). KIR mRNA and surface protein expression

Receptor transcripts and surface expression were quantified by RQ-PCR and flow cytometry, respectively. Patients with positive MRD had a higher mRNA level of Tel B- associated KIR2DS1 (p=0.022) and a lower level of Tel ^-associated KIR3DL1 (P=0.007) and KIR2D transcripts (p=0.01 1 ) than did those with negative MRD (Table 5). Table 5. Comparison of NK cell receptor mRNA transcript level and MRD

Mean (SD) copies x 10 ^J per pg of RNA

Receptor All patients Negative MRD Positive MRD p value

KIR2DL1 44.6 (82.9) 48.9 (87.6) 16.4 (27.4) 0.011

KIR2DL2 19.1 (61.1) 20.9 (65.1) 7.6 (16.4) 0.21

KIR2DL3 51.3 (71.3) 54.3 (74.5) 31.5 (42) 0.11

KIR2DL4 26 (44.3) 24.5 (40.8) 35.9 (64.2) 0.19

KIR3DL1 75.8 (101.9) 83.6 (106.9) 23.8 (24.2) 0.007

KIR3DL2 1 ,388.3 (11 ,034.3) 1 ,547.4 (11 ,834.6) 335.8 (935.3) 0.14

KIR2DS1 8.2 (18.6) 7.8 (19.2) 11.4 (14.6) 0.022

KIR2DS2 37.5 (123.1) 35.5 (120.7) 50.4 (143.2) 0.36

KIR2DS3 4.8 (19.7) 5.2 (21) 2.5 (5.2) 0.96

KIR2DS4 137.2 (400.1) 137.8 (402.5) 133.3 (399.4) 0.3

KIR2DS5 4.3 (10.7) 4.4 (11.2) 3.9 (6.8) 0.63

KIR3DS1 19.8 (48) 17.4 (45.2) 36 (63.3) 0.074

NKp30 65.5 (87.1) 60.8 (68.6) 96.5 (165.5) 0.77

NKp44 1.1 (3.9) 1 (3.4) 1.8 (6.2) 0.29

NKp46 61.3 (80) 65.5 (84.3) 33.8 (32) 0.26

NKG2D 4,033.8 (17,096.3) 4,155.9 (18,124.6) 3,226.5 (7,698.3) 0.36

The association of MRD with surface expression of KIR2DL1, KIR2DL2/3, and KIR3DL1 as enumerated by flow cytometry was analyzed in the context of the presence of their corresponding ligand. The odds of being MRD positive in a patient with HLAC2 ligand increased by 2.01-fold (95% CI 1.05-3.85) for every percentage increase in NK cells expressing KIR2DL1 on their surface (p = 0.034). There were no significant association between receptor and ligand for KIR2DL2, KIR2DL3 and KIR3DL1.

Other NK cell receptor surface expression

The proportion of NK cells expressing NKG2A, NKp46, and FAS receptor were significantly associated with the MRD status. The NK cells from the MRD positive patients had lower frequency of expression of NKG2a (mean + SD, 27.1 % ± 16.1 % vs. 38.3 ± 14.3%, p = 0.0012) and NKp46 (37.2% ± 37% vs. 56.6% ± 29%, p=0.0074), but higher expression of FAS (44.8% ± 43.2% vs. 23.8% ± 31.8%, p=0.032) than NK cells from MRD negative patients. In terms of surface density, the mean florescence intensity of FASL was lower in the MRD positive group than the MRD negative group (348 ± 201 .3 vs. 543.1 ± 358.7, p=0.029).

Leukemia Blast Characteristics

Among the 13 biomarkers, ICAM, PI-9, and NTBA were statistically associated with MRD status. The leukemia blasts from the MRD positive patients had a greater frequency of expression of ICAM (mean +SD, 34.9% ±30.2% vs. 22.5% ±24.5%, p=0.035) and PI-9 (mean +SD, 63.3% ± 35% vs. 45.6% ±35.2%, p=0.027) than blasts in the MRD negative group. Further, the amount of PI-9 in the MRD positive group was greater than that in the MRD negative group (MFI mean +SD, 996 ± 677.8 vs. 691 .6 ± 527.5, p=0.038). The amount of NTB-A expressed on the blasts was also greater in the MRD positive group (MFI mean +SD, 235.9 ±186.5 vs. 184.1 ± 79.4, p=0.04).

Predictive Model

A 5-parameter predictive model and its receiver operating characteristics (ROC) curve were created based on the most significant results of immunophenotyping and KIR genotyping, including PI-9, FasL, Granzyme B, NKp46 and KIR2DL5A. The probability of positive MRD ranged from as low as 3.32 * 10 ^"9 % to as high as 81.6% (Table 6), using cutoffs which were established statistically from individual ROC (>650, 415, 4070, and 30 for the first four predictors, respectively, and the presence of KIR2DL5A). The 5- biomarker composite model had a sensitivity of 100% and specificity of 80% in predicting MRD (Figure 2); the AUC of its ROC curve improved to 0.93 when compared with those of individual biomarker (all AUC <0.67). Table 6. Probability of positive MRD at the end of induction based on five markers.

DISCUSSION

The activity of NK cells is regulated by the engagement of their inhibitory and activating surface receptors with ligands on the leukemia lymphoblasts. Cytotoxicity is achieved through perforin/granzymes or FasL ^3,19 . Herein, we presented our

comprehensive evaluation of these molecules in NK cells and target leukemia cells. A MRD predictive model was developed that would be expedient for clinical use before induction chemotherapy.

KIR diversity is generated through haplotype gene content on chromosome 19q13.4, allele polymorphism, and stochastic surface expression ^20"23 . In our study, the presence of genes KIR2DL5A, KIR2DS1, and KIR2DS3 were significantly associated with a positive MRD. Interestingly, these three genes are exclusively found in group-B KIR haplotypes, suggesting that B haplotypes are associated with a poor induction response in pediatric ALL. When we compared the haplotypes A/A versus A/B directly, the telomeric rather than the centromeric B motifs appeared to be the key determinant, as the odds of being MRD positive with Tel-A/B genotypes was significantly higher than that of Tel-A/A, whereas no difference was observed between Cen-A/B and Cen-A/A. In contrast to our study, some investigators have found that the group-B KIR haplotypes were associated with improved overall survival and event-free survival, as well as lower transplant-related mortality ^24"28 . However, others corroborate our findings of an A haplotype benefit and a B haplotype disadvantage ^29,30 . The A haplotypes were associated with a complete molecular response in chronic myeloid leukemia ³¹ and an improved disease free survival in patients receiving a haploidentical T-cell depleted stem cell transplant for leukemia. The risk of relapse in the latter study increased with the number of activating donor KIR genes present ³² . Because B haplotypes typically contain larger numbers of activating KIR genes than the A haplotypes (which contain only two activating genes KIR2DL4 and KIR2DS4 that are often disrupted by mutations rendering them non-functional) ^33"36 , these data collectively suggest that the risk of relapse might be dependent on the activating gene content in the B haplotypes. In this regard, NK cells possessing Tel-B associated KIR2DS1 have been shown to be hypo-responsive because of tolerance induced by C2 ligands ³⁷ ' ³⁸ .

To further dissect the role of activating KIRs from that of inhibitory KIRs in MRD, we directly measured the patients' KIR repertoire by RQ-PCR. This approach had two advantages. First, the RQ-PCR allowed us to analyze all 15 family members of KIRs individually, as monoclonal antibodies for flow cytometry are either not available or cannot discern the activating versus inhibitory KIRs. Second, previous studies have shown that genotypes do not always correlate with phenotypes ^39,40 . In our cohort, the MRD positive group had more mRNA transcripts of the B-haplotypes associated KIR2DS1 activating receptor than the MRD negative group, collaborating with our genotype findings of detrimental Tel-B effect. By contrast, the MRD negative group expressed more mRNA of inhibitory receptors KIR2DL1 and A-haplotypes associated inhibitory KIR3DL1, supporting the hypothesis of the importance of inhibitory KIR acquisition during NK cell licensing for missing-self recognition ^41"43 . The licensing or arming model proposes that inhibitory receptor and MHC ligand interactions educate the NK cells to kill when it encounters a target cell missing the cognate ligand ^44"47 . The greater the number of inhibitory receptors on NK cells, the stronger the cytotoxic responsiveness ^48,49 . The absence of these inhibitory receptors, even in the presence of activating receptors, leaves the NK cell hypo-responsive. Alternatively, our Tel-B genotype and RQ-PCR data also supports the disarming model, which states that NK cells exhibiting more stimulatory signal become dampened overtime ^50,51 . In our patients, those with a poor response to induction chemotherapy are typified by the Tel-B genotypes with high level of expression of activating KIR2DS1 and low level of expression of inhibitory KIR2DL1 and KIR3DL1, a phenotype found in immature NK cells and unlicensed NK cells ^52,53 . In fact, KIR2DL5A is the most significant risk factor for MRD. Although the function and ligand of KIR2DL5A are unknown, this KIR gene is almost in complete linkage disequilibrium with the absence of Te\-KIR3DL1 and the presence of Te\-KIR2DS1, KIR2DS3, KIR2DS5, and KIR3DS1; NK cells of these genotypes (more activating and less inhibitory gene content) would be predictably hypo-responsive based on the arming and disarming model ^50,51 .

In addition to KIRs, other NK cell receptors and effector molecules are crucial determinants of antileukemia response ^3,10,19,54 . A NCR ^du " phenotype, for example, has been associated with poor leukemia control in adult AML ^4,5 , in line with our data showing that MRD was associated with low expression of NKp46. Furthermore, the amount of FasL in the NK cells of the MRD positive group was smaller than that of the MRD negative group; FasL is essential to induce the death pathway in the target cells ⁵⁵ .

The leukemia cell-intrinsic factors may also play a role in NK-cell escape. In our patients, MRD positivity was associated with the presence of C2-ligand and the abundance of intracellular PI-9. While C2 may inhibit KIR2DL1 , PI9 can resist granzyme B ^56,57 or FasL mediated cytotoxicity, rendering both cytotoxic pathways ineffective ⁵⁸ .

Post-induction MRD has been established to be one of the most important prognostic markers for childhood ALL and MRD-based risk-adaptive continuation regimens have been shown to improve patient outcomes ⁵⁹ . Using five biomarkers associated with the NK-specific pathways identified in this study, we create a novel MRD- relevant predictive model that can be used at diagnosis so that risk-adaptive therapy can be implemented before (rather than after) induction chemotherapy. For instance, patients in groups 1-16 as listed in Table 4 had a <1x10 ^"5 % risk of positive MRD; these patients would have an excellent outcome when treated with regimens similar to that of TotalXVI By contrast, alternative therapy should be considered for patients in groups 17-25 who had an intermediate risk ranging from 0.2% to 10%, and especially for those in the high risk groups with a risk ranging from 13.3% to 81.6%. The laboratory tests for the five biomarkers can be completed within 24 hours and the predictive model can be implemented at diagnosis with favorable operating characteristics: 100% sensitivity and 80% specificity, corresponding with a 100% negative predictive value.

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All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.